User:Nadia Dorochko

UVM, Biochemistry Ph.D student

Factor IX
 Factor IX (plasma thromboplastin component, Christmas factor, or hemophilia B factor) is a single-chain vitamin K-dependent procoagulant glycoprotein. It is synthesized by the liver hepatocyte as a pre-prozymogen that requires extensive posttranslational modification. The pre-prozymogen contains a pre-pro sequence that is followed by a polypeptide region. The pre-peptide is a hydrophobic signal peptide at its amino terminal that transports the growing polypeptide into the lumen of the Endoplasmic Reticulum. Once inside the ER, this signal peptide is cleaved by signal peptidase. The pro-peptide contained in the protein induces the docking of the polypeptide to the vitamin K-dependent carboxylase (γ-glutamyl carboxylase), where is modified by γ-carboxylation. The posttranslational modification creates a fully gamma-carboxylated mature zymogen which can now associate with anionic phospholipid surface.

The structure consists of 415 amino acides that are separated into an amino terminal Gla domain (12 Gla residues), which is a characteristic feature of all vitamin K–dependent factors. The Gla region is followed bt two tandem EGF domains (residues 85-127), activation peptide region which is cleaved off upon activation of FIX to FIXa, and a serine protease domain, which contains enzymatic activity.

Before protein is secreted into the circulation it undergoes posttranslational modifications, which include gamma-carboxylation, beta-hydroxylation, N and O-linked glycosylation, sulfation, and phosphorylation. Gamma-carboxylation is a vitamin K–dependent process in which the gamma-carboxylation of the glutamic acid residues forms gamma-carboxyglutamyl (Gla) residues in the mature protein and requires reduced vitamin K, oxygen, and carbon dioxide to perform its functions (I will give you an image later). In this modification the negative charge allows the FIX to undergo a conformational change in order to interact with Ca2+ and interact with the anionic phospholipid membrane. In the absence of calcium the Gla residues are exposed to the solvent, however upon its binding the protein exposes its hydrophobic residues which are inserted into the lipid membrane.

Hydroxylation occurs in the EGF-1 domain of FIX on aspartic acid residues to form a erythro-β-hydroxy aspartic acid (HYA). The addition of a hydroxyl group (OH) to aspartic acid involves the recognition of a Cys-X-Asp-X-X-X-X-Tyr/Phe-X-Cys-X-Cys consensus sequence (reference 334).

Glycosylation involves the addition of a carbohydrate in the activation peptide of FIX. The activation peptide is cleaved off at residues R145-A146 and R180-V181, thus converting the zymogen to an active form.

The serine protease domain of FIX accounts for half of its mass and contains a conserved catalytic triad made of Asp, His, and Ser. The binding pockets of these vitamin-K depended proteases recognize a small number of amino acids sequences allowing them to cleave at arginyl residues with high substrate specificity. Serine’s hydroxyl group carries out the nucleophillic attack. While the imidazol ring of hisidine takes up the liberated proton and the carboxylate ion of Asp stabilizes the developing charge.

Deficiency of this glycoprotein leads to hemophilia B, a common X linked inherited coagulation disorder that affects 1 in 30, 000 males (reference 458, 464). Females are rarely affected by this disorder unless there is a mutation in the FIX gene (465, 466). The mutations that have been identified in the factor IX gene and lead to proteins reduced activity include; large and small deletions, point mutations, and missense mutations.

Pre-prozymogen Image


Major Components of the FIX Structure

Exon

^ - Epidermal growth factor 1

^^ - Epidermal growth factor 2

pro-peptide

Gia domain

EGF1